1. Field of the Invention
The present device relates to analytic methods and devices for separation and quantification. More particularly, the present invention relates to the separation and quantification of selenium containing proteins and devices that support the process of separation and quantification.
2. Description of the Related Art
There are a number of essential micronutrients in both humans and animals, and many cases their significance is not fully understood. For example, selenium (Se), which is found in both humans and animals in almost trace amounts, can be used as an accurate health marker. Lower than normal levels of Se in humans can affect reproduction, immuno-response, miscarriage rate and thyroid function (to name just a few areas), as well as suppression of the destruction of free radicals that may be responsible for the formation of certain types of cancer. Typically, elements such as Se are found within a human or animal body in proteins, which in this case are referred to as, for example, selenoamino acids (selenocysteine and selenomethionine).
There are several studies that support the finding that the measurement of selenium-containing proteins are better health markers than a “total” selenium count. In fact, the main selenium-containing proteins found in human serum and plasma are albumin, gluthathione peroxidase (GSHPx) and selenoprotein P (SepP), see Arce-Osuna, M., DISSERTATION: ANALYTICAL METHOD DEVELOPMENT FOR SELENIUN-CONTAINING PROTEINS OF CLINICAL INTEREST, University of Massachusetts, Analytical Chemistry Department, Amherst Ma, September 2005, incorporated herein by reference.
The most common techniques used for the separation, identification and quantification of selenium containing proteins are mainly based on antibody specificity and affinity chromatographic processes. For example, to separate the selenium-containing protein, techniques such as immunoassay, anion exchange chromatography, heparin affinity chromatography, immobilized metal affinity chromatography (IMAC) and size exclusion (SEC) have been used in the art with some degree of success.
In addition, protein identification has been accomplished by a combination of chromatographic retention time, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and GSHPx activity of chromatographic fractions.
Quantification of the amount of selenium (as well as sulfur) in a sample has been performed by using inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), and by derivatization for fluorimetric detection. However, some of the major disadvantages of the aforementioned methods include poor reproducibility, specificity, inadequate sensitivity, analyte losses, and contamination.
In order to determine the amount of selenium in each protein, there are salts contained within the mobile phases (buffers) for affinity chromatography, and organic solvents used in reversed phased chromatography pose a problem when a coupled liquid chromatograph inductively coupled plasma mass spectrometry (LC-ICP-MS) instrument is used. More specifically, the high concentration of salts (above 0.5 to 1.0 g/mL−1) in ICP-MS, in most cases, has a detrimental effect on the analyte signal because of matrix effects. In addition, the organic solvent that is typically used in chromatography is also a problem in ICP-MS measurements. There is a problem in that the high vapor pressure in the spray chamber reduces the analyte transport to the plasma torch, and at high concentrations, reduces the effective ionization power of the plasma. The results is a significant ICP-MS background drift resulting from a chromatographic gradient run. Sending organic solvents into an ICP-MS may also cause the build-up of carbon in the sampling cones. FIG. 1 is a photo showing some of the carbon build-up that can occur.
Moreover, the problems encountered when an inductively coupled plasma mass spectrometry (ICP-MS) unit was coupled directly to the continuous flow from reversed phase liquid chromatograph includes a high background signal, first increasing and then decreasing in magnitude during the course of a chromatographic gradient solvent, as well as the aforementioned carbon build-up and transport difficulties.
There have been attempts to reduce the concentration of the organic solvent that reaches the ICP-MS and/or reduce the amount of effluent that arrives at the ICP-MS. It is, for example, a common practice to add a small amount of oxygen to a nebuliser gas flow and to operate the plasma at high power in order to make the system more robust to the effects of organic solvent.
For example, desolvated aerosol has been produced in a number of different ways, such as combining thermospray or ultrasonic nebulisers with a membrane desolvator, or connecting a membrane with cryogenic cooling. However, these approaches still result in a low concentration of organic solvent being introduced into the ICP. Therefore, it is still very difficult to eliminate residual solvent from an ICP spray chamber after each chromatographic run, such that a subsequent chromatographic run is affected by the previous run, having an affect on the integrity of the measurements.
Accordingly, research conclusions might be affected by the uncertainty or the bias of the methods used, and there is a need in the art for a more accurate quantification of both selenium and selenium-containing protein.